The present invention relates to a non-human animal model for obesity and uses of such an animal for studying and developing methods for identifying compounds for use in the regulation of insulin resistance in obesity and type II diabetes, as well as a method of treating insulin resistance in obesity and type II diabetes by administration of such compounds.
Diabetes, and conditions related thereto, are major health concerns throughout the world, and, particularly in the United States, contribute to morbidity and mortality. Non-insulin dependent diabetes mellitus (NIDDM), also known as type II diabetes, is the major form of diabetes in developed countries. While a large number of environmental and genetic factors contribute to the risk of NIDDM in the United States, prolonged obesity is by far the largest risk factor. The molecular basis of this association, however, is not fully understood. As a consequence, efficient means of therapeutical intervention are lacking.
Before the development of diabetes, many obese patients develop a peripheral resistance to the actions of insulin. The molecular basis of insulin-resistance in obesity has been the subject of intensive study, by nonetheless remains elusive. Insights into components and mechanisms of the link between obesity and insulin resistance have been gained from mouse models of obesity which display obesity-induced insulin resistance. The molecular basis of the various mouse obesity models covers a range of mechanisms; nonetheless these all develop diabetes, either before or after the onset of obesity.
Obesity in humans and rodents is commonly associated with insulin resistance, (i.e., smaller than expected responses to a given dose of insulin) (LeRoith et al., Diabetes Mellitus: a Fundamental and Clinical Text. (Lippincott-Raven, Philadelphia, 1996); DeFronzo et al., Diabetes Care 15:318-68 (1992); Rifkin et al., Diabetes Mellitus, (Elsevier, N.Y., 1990)). The mechanisms linking obesity and insulin resistance are not known. Studies on the potential mechanistic basis of obesity-induced insulin resistance have revealed numerous potential sites, making a single basic mechanism for explaining insulin insensitivity unlikely (Rifkin et al., Diabetes Mellitus, (Elsevier, N.Y., 1990)). Both insulin secretion and action can be impaired. Accordingly, sites at the anatomical, cellular, and molecular level are the xcex2-cells of the pancreas, and membrane carriers and enzymes regulating metabolic pathways in liver, fat, and muscle. An example for impaired insulin secretion can be found in a rodent model of obesity with non-insulin-dependent diabetes mellitus, the Zucker diabetic fatty (fa/fa) rat, where overaccumulation of triglycerides in the pancreatic islets leads to gradual depletion of xcex2 cells (Lee et al., Proc Natl Acad Sci USA 91:10878-82 (1994); Shimabukuro et al., Proc Natl Acad Sci USA 95:2498-502 (1998)). Insulin action can be impaired in a number of ways, involving insulin sensitive carriers or pathways, or the insulin receptor directly. Earlier studies indicated that quantitative regulation of the insulin sensitive glucose transporters (Glut-4) may contribute to insulin resistance; however, this factor alone is probably inadequate to explain the extent of insulin resistance. For instance, mutant mice lacking Glut-4 develop only mild hyperinsulinemia (Katz et al., Nature 377:151-5 (1995)). More recently studies have focused on defects at the level of the insulin receptors themselves and at post-receptor events in type 2 diabetes, specifically the intrinsic catalytic activity of the insulin receptor and downstream signaling events. A reduction in tyrosine phosphorylation of both the insulin receptor (IR) and the insulin receptor substrate-1 (IRS-1) has been noted in both animals and humans with type 2 diabetes (Le Marchand-Brustel et al., J Recept Signal Transduct Res 19:217-28 (1999)). Importantly, this occurs in all of the major insulin-sensitive tissues, namely the muscle, fat and liver. Disruption of IRS-2 in mice impairs both peripheral insulin signaling and pancreatic xcex2-cell function (Withers et al., Nature 391:900-4 (1998)). Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was found to be profoundly affected in response to insulin (Kerouz et al., J Clin Invest 100:3164-72 (1997)). The regulation of gene expression by insulin in the liver is impaired for the genes for glucokinase and phosphoenolpyruvate carboxykinase (PEPCK) (Friedman et al., J Biol Chem 272:31475-81 (1997)). A modulator of insulin action is tumor necrosis factor (TNF)-xcex1, which blocks insulin through its ability to inhibit insulin receptor tyrosine kinase activity (Feinstein et al., J Biol Chem 268:26055-8 (1993)). Mice lacking TNF-xcex1 function are protected from obesity-induced insulin resistance (Uysal et al., Nature 389:610-4 (1997)). Another modulator of insulin sensitivity is protein tyrosine phosphatase-1B (PTP-1B) which acts as a negative regulator of insulin signaling (Cicirelli et al., Proc Natl Acad Sci USA 87:5514-8 (1990)). Mice deficient in PTP-1B are interestingly more sensitive to insulin but resistant to obesity (Elchebly et al., Science 283:1544-8 (1999)). Most recent studies have focused on the peroxisome proliferator-activated receptor xcex3 (PPARxcex3), a member of the nuclear-hormone-receptor family (Auwerx, Diabetologia 42:1033-49 (1999)). Mutations in humans of PPARxcex3 suggest that this molecule is required for normal insulin sensitivity in humans (Barroso et al., Nature 402:880-3 (1999)). It is not clear at the moment whether insulin resistance in human obesity might result from impaired PPARxcex3 signaling. What is now clear is that decreased signaling capacity of the insulin receptor can be an important component of obesity-induced insulin resistance.
At the intracellular, metabolic enzyme, level, insulin-resistance in obesity seems to consist of increased activities of key enzymes of pathways known to be stimulated by insulin (i.e. glycolysis, lipogenesis), but also of increased activities of key enzymes of pathways normally depressed by insulin (Belfiore et al., Int J Obes 3:301-23 (1979)). This failure of insulin to depress enzymes of catabolic pathways manifests itself in enhanced basal lipolysis in adipose tissue, increased amino acid release from muscle, and elevation in the activity of key gluconeogenic enzymes in the liver.
As mentioned above, there are a number of mouse models with genetic obesity-diabetes syndromes (Herberg, et al., Metabolism 26:59-99 (1977)). They characteristically have hyperglycemia, hyperinsulinemia, and obesity, albeit to different degrees, with different times of onset, and for different reasons. In the yellow obese mouse (Ay/a) a dominant mutation of the agouti locus causes the ectopic, ubiquitous expression of the agouti protein, resulting in a condition similar to adult-onset obesity and non-insulin-dependent diabetes mellitus (Michaud et al., Proc Natl Acad Sci U S A 91:2562-6 (1994)). Obese (ob/ob) (Zhang et al., Nature 372:425-32 (1994)), diabetes (db/db) (Tartaglia et al., Cell 83:1263-71 (1995)), fat (cpe/cpe) (Naggert et al., Nat Genet 10:135-42 (1995)) and tubby (tub/tub) (Kleyn et al., Cell 85:281-90 (1996); Noben-Trauth et al., Nature 380:534-8 (1996)) are mutations in single recessive genes, specifically in the genes for leptin, the leptin receptor, carboxypeptidase E, and a member of a new family of genes encoding tubby-like proteins, respectively. Obese mice have a diabetes-like syndrome of hyperglycemia, glucose intolerance, and elevated plasma insulin. The diabetes syndrome develops after the onset of obesity, and is probably the result of it. In diabetes mice elevation of plasma insulin at 2 weeks of age precedes the onset of obesity at 3-4 weeks; blood glucose levels are elevated at 4-8 weeks. Fat mice have hyperinsulinemia consistent throughout life in association with hypertrophy and hyperplasia of the islets of Langerhans; hyperglycemia is transient. In tubby mice, plasma insulin is increased prior to obvious signs of obesity, and islets of Langerhans are enlarged; here blood glucose is normal.
As discussed above, the molecular basis for insulin resistance in obesity is unknown. Increased leptin levels cannot account for this, since insulin resistance occurs in the leptin deficient ob/ob mutants. Therefore, there must be some other molecular xe2x80x9csignalxe2x80x9d in obesity which mediates the insulin-resistance seen in obesity.
Faced with such a long felt, but unsolved need for simple and effective methods to prevent or reduce the negative effects of diabetes, researchers, over the last several decades, have expended literally hundreds of millions of dollars to investigate compounds that can be used to treat and/or prevent diabetes. While altering glucose can affect the occurrence and the severity of diabetes, so can the regulation of insulin resistance in obesity. This latter approach has been an under-appreciated field relative to diabetes. The present invention is directed to the prevention and/or treatment of diabetes through the regulation of insulin resistance in obesity.
One embodiment of the present invention relates to a method to identify compounds useful in regulating insulin resistance in obesity and type II diabetes. This method includes the steps of: (a) administering a compound having melanocyte stimulating hormone (MSH) biological activity to a genetically modified non-human animal comprising a genetic modification within two alleles of its Pomc locus, wherein the genetic modification results in an absence of proopiomelanocortin (Pomc) peptide activity in the animal, and wherein administration of the compound having MSH activity induces insulin resistance in the animal; (b) administering a compound to be evaluated to the non-human animal model; and, (c) selecting compounds from (b) that decrease the insulin resistance in the non-human animal as compared to in the absence of the compound of (b).
In one embodiment, the genetic modification is selected from the group consisting of a deletion, an insertion, a substitution and an inversion of nucleotides in the Pomc locus. In another embodiment, the genetic modification is a deletion of a nucleic acid sequence within two alleles of the Pomc locus, wherein the deletion results in an absence of expression of Pomc peptides by the animal. In another embodiment, the genetic modification is a deletion of a nucleic acid sequence comprising exon 3 of Pomc or a portion of exon 3 of Pomc sufficient to prevent expression of Pomc peptides by two alleles of the Pomc locus. In another embodiment, the animal is a mouse, and wherein the genetic modification is a deletion from the genome of exon 3 of Pomc (SEQ ID NO:7).
In one aspect, the compound having MSH biological activity in step (a) is selected from the group consisting of: MSH, a biologically active fragment of MSH, a homologue of MSH, a peptide mimetic of MSH, a non-peptide mimetic of MSH, and a fusion protein comprising an MSH protein or fragment thereof. In another aspect, the compound of (a) having MSH biological activity is xcex1-MSH.
In one aspect, the compound of (b) to be evaluated is an antagonist of MSH biological activity. In another aspect, the compound of (b) to be evaluated is administered prior to the step of administering the compound of (a) having MSH biological activity.
Yet another embodiment of the present invention relates to a method to decrease insulin resistance in a mammal, comprising administering to the mammal that has insulin resistance a therapeutic composition comprising an antagonist of melanocortin stimulating hormone (MSH) biological activity, wherein the antagonist decreases insulin resistance in the mammal. In one aspect, the antagonist of melanocortin stimulating hormone (MSH) is selected from the group consisting of a fragment of MSH having MSH antagonist action, a homologue of MSH having MSH antagonist action, a peptide mimetic of MSH having MSH antagonist action, a non-peptide mimetic of MSH having MSH antagonist action, and a fusion protein comprising any of the MSH antagonist compounds. In another aspect, the antagonist of MSH is a soluble MSH receptor or fragment thereof that binds MSH. In yet another aspect, the antagonist of MSH is an antibody that selectively binds to MSH and thereby reduces or blocks the activity of MSH. In another aspect, the antagonist of MSH is an antibody that selectively binds to a receptor for MSH and reduces or blocks the ability of MSH to bind to the receptor.
The therapeutic composition can be administered by any suitable route, including, but not limited to, transdermally, topically, and parenterally. In one aspect, the therapeutic composition is administered in a controlled release formulation. In one aspect, the MSH antagonist is administered in a dose of from about 0.1 xcexcg to about 10 mg per kg body weight of the animal.
Another embodiment of the present invention relates to a method to treat diabetes associated with insulin resistance in a mammal, comprising administering to the mammal that has insulin resistance and diabetes a therapeutic composition comprising an antagonist of melanocortin stimulating hormone (MSH) biological activity, wherein the antagonist decreases insulin resistance in the mammal.